Patients with cystic fibrosis (CF) could potentially be treated using their own stem cells that have been manipulated by gene therapy, suggests a study reported in this weeks Proceedings of the National Academy of Sciences (PNAS). The authors, who represent five institutions, including the University of Pittsburgh School of Medicine and Childrens Hospital of Pittsburgh, demonstrate for the first time that human bone marrow-derived adult stem cells can be coaxed to differentiate into airway epithelial cells and that encoding these cells with the gene that is defective in CF restores an important cellular function essential for keeping the airways clear of mucus and air-borne irritants.

"Our results provide proof of principle that a cell-based therapy using marrow stromal stem cells is both a feasible and promising clinical approach. We plan to further investigate its potential and are hopeful that we can perform a small clinical trial within the next two to three years," noted senior author Jay K. Kolls, M.D., professor of pediatrics, immunology and molecular genetics and biochemistry at the University of Pittsburgh School of Medicine and chief, Division of Pediatric Pulmonology and Laboratory of Lung Immunolgy and Host Defense at Childrens Hospital of Pittsburgh.

CF is the most common, fatal genetic disorder affecting Caucasians and is characterized by a mutation of a gene that sits on the surface of epithelial cells, including those that line the airways in the lungs. The gene, transmembrane conductance regulator (CFTR), is responsible for channeling chloride out of cells, an essential function that influences the level of airway surface liquid. This fluid sits atop the airway cells and provides support for the hair-like projections called cilia that sweep mucus and dirt away from the cell. When the level of liquid is too low, as is the case in CF, mucus builds up and the area becomes an attractive environment for bacteria to colonize and cause infection.

Finding ways to correct the gene defect responsible for CF has been the focus of much research and some clinical studies are even looking at gene therapy approaches. To date, no studies have evaluated the potential of adult stem cells or the use of stem cells in combination with ex vivo gene therapy, whereby the CFTR gene is delivered to the cells in a laboratory setting before their introduction into the patient.

While some studies have suggested the potential for bone marrow derived stem cells to differentiate into airway epithelial cells, Dr. Kolls and his co-authors have provided significant proof. Using a method they developed and report in PNAS, stem cells taken from normal subjects and placed in a culture with mature epithelial cells for 14 days differentiated into the specialized cells, taking on their shape and displaying their characteristic protein markers. Importantly, stem cells aspirated from the bone marrow of three CF patients and co-cultured in the same manner also produced airway epithelial cells, but, as expected, were deficient for the CFTR gene. To correct the genetic flaw, the researchers used the Maloney murine leukemia virus as a vector to shuttle the CFTR gene into the stem cells.

A technique called polymerase chain reaction, or PCR, confirmed the gene was expressed in the newly differentiated airway epithelial cells, reported Guoshun Wang, M.D., D.D.M, of Louisiana State University (LSU), where Dr. Kolls previously had worked, and Bruce A. Bunnell, Ph.D, of Tulane University, the studys first authors. Moreover, the gene therapy-treated cells from CF patients had the same developmental potential as bone marrow-derived stem cells from healthy people.

To determine whether the gene therapy actually corrected the CFTR defect, the researchers exposed the cells to a medium containing labeled chloride and a molecule that regulates the intake and secretion of salts and water necessary for cellular function. If the CFTR gene were not functioning properly, the chloride taken in by the cell would not be able to be secreted. Yet, as the authors reported, the CFTR-corrected stem cells secreted significantly more chloride than stem cells from CF patients without the inserted gene.

"Being able to demonstrate that the gene permitted enhanced chloride channeling was critical to determining the potential of the stem cell-based therapy in the clinical setting," said Dr. Kolls.

Although he acknowledges that more work needs to be conducted, including developing a way to identify the specific subpopulation of bone marrow stem cells that are key to successful engraftment, Dr. Kolls is optimistic about the possibility that a patients own stem cells – which would not be subject to immune system attack – could be manipulated by gene therapy and used successfully as a treatment for CF. The ex vivo approach to gene therapy may offer several advantages, including the ability to screen the cells before actually delivering them to patients. In addition, the virus vector being studied by Dr. Kolls is bigger than others being used in trials and has the capacity for both the gene and its accessory apparatus, which may help ensure long-term function.

To further study the potential of the stem cell approach, including in CF patients, Dr. Kolls and colleagues from Childrens Hospital, in collaboration with the University of Pittsburgh, are seeking funding from the National Institutes of Health (NIH) to establish a center focusing on the development of adult stem cell-based therapies for diseases affecting the lung, heart and blood vessels.

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